PROJECT SUMMARY Our Wellstone research has identified two classes of FSHD therapeutics for pre-clinical in vivo testing development. The goal of Project 3 is to validate the in vivo efficacy of these candidate therapeutics and/or related compounds for IND enabling studies within the next five years. Studies will be conducted in collaboration with industry partners with expertise and successful track records in drug development and with resources to support the proposed research strategies. One class of FSHD drugs to be investigated is based on the DUX4 morpholino, FM10, which Wellstone research has previously established targets the DUX4 4qA polyadenylation sequence and knocks down DUX4 transcriptional activity in FSHD patient cells and in FSHD xenograft muscle. RNA-Seq studies further established that FM10 does not have significant off-target effects, but is effective at knocking down expression of the entire suite of DUX4 target genes. Studies in Specific Aim 1 focus on in vivo somatic delivery and therapeutic potency of the FM10 sequence for DUX4 knockdown in FSHD xenograft muscle, using unique, non-toxic cell-penetrating peptides developed and validated by Sarepta Therapeutics. Additional PPMOs targeting DUX4 will be identified in the FM10 sequence region using FSHD primary patient myogenic cells to optimize therapeutic response and RNA-Seq to evaluate their on- and off-target activities. A second class of FSHD drugs to be investigated are 4-methylumbelliferone (4-MU) and other inhibitors of the hyaluronic acid (HA) signaling pathway. Wellstone research has shown that 4-MU is a potent inhibitor of DUX4 toxicity and other DUX4 molecular pathologies, which suggests that the HA signaling pathway plays an essential role in DUX4-dependent pathology, likely through a mechanism involving C1QBP, a protein that binds to both DUX4 and HA. This identifies the HA pathway as a target for additional FSHD therapeutic development, and also suggests that 4-MU, already in use as an antispasmodic therapeutic and extensively studied in mice for its anti-cancer and anti-inflammation properties, is a promising FSHD drug for in vivo testing. Specific Aims 1 and 2 focus on investigations of the functions of C1QBP in DUX4 pathology and the identification and testing of signaling pathway inhibitors that target this pathway to block DUX4 toxicity. 4-MU will be examined on a global level to identify its full range of on- and off-target effects, and the HA pathway will be studied using specific pathway inhibitors to identify additional targets of HA signaling for therapeutic investigation. HA pathway inhibitor studies will be conducted in collaboration with Genea Biocells, who will provide clinically validated HS signaling pathway inhibitors as well as industry expertise and experience in drug development. Drugs with high potency for inhibiting DUX4 toxicity, and low toxicity and off target disruptions, will be identified for in vivo activity, alone and in combination with 4-MU, in the Tamoxifen-Inducible Cre (TIC) DUX4 mouse model and, if suitable, in the FSHD xenograft muscle model.